Human Practices

Here, we present an overview of the sample characteristics from our survey in terms of demographic background:

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Alongside that, we performed a Latent Class Analysis (LCA) to explore differing attitudes and expertise levels regarding the progRAM project and RNA technologies. The LCA is a statistical method used to uncover hidden subgroups (latent classes) within a population by analyzing patterns of responses across multiple observed variables (Weller et al., 2020). Through this approach, the population was divided into three distinct latent classes, which are displayed in the graph below.

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​ The graph illustrates how each class responded to variables such as beliefs about RNA safety, the potential of the progRAM project to advance research, familiarity with molecular biology, and the perceived need for regulations. Particularly, we found it interesting that the belief that RNA-based molecular recording technologies are safer than DNA-based ones is prevalent among all three classes, and that the need for additional regulatory measures and the belief on widespread use of progRAM are shared between classes 1 and 3, which comprise most of our respondents.

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​ Additionally, most concerns were fairly consistent across our sample population, sharing common worries about off-targets, potential misuse, and the practical challenges of RNA-based molecular recording technologies. And even while RNA-based editing offers a safer, more transient alternative to DNA modification, it still carries inherent risks related to efficiency and precision, which we have carefully incorporated into our design. Even though we are a foundational project, you can explore how we address issues like RNA stability, specificity, and safety in the context of our design and envisioned applications on our Description and Engineering pages.

Finally, as strong advocates of openness and transparency in research, we are also committed to sharing the full database and codebook of our results, along with a template of the survey for further use and adaptation, which you can access by clicking the buttons below:

Our survey taught us a lot about how our RNA-based molecular recording system was received and its potential impact. We shared and discussed these findings through social media posts to spark constructive discussions on potential improvements and applications beyond our academic interactions.

The insights we gained were invaluable, guiding us in our internal strategies and enhancing our outreach efforts. The results were presented as crucial input in our meetings with experts, and they were presented in events like our Panel Discussion and our upcoming TUM Open Day to create a starting point for a broader conversation on the impact of our project.

Initial State of the Project

Summary: We had already done research sessions on molecular recording and the use of gRNAs, exploring different systems and novel readout methods that were discussed with our PI. We designed 30 and 50 bp tapes to test based on the optimal lengths described in literature. Here, the RNA stability of our tapes already was considered a major necessity, as systems without capping were not suitable in cellular environments. To that end, we had been evaluating the feasibility of alternatives like the circularization of RNA with the TORNADO system, integration of WPRE elements, and the use of IRES, among many options that were considered throughout the project. With this, we also constructed a minimal vector to simplify the experimental construct and remove unnecessary restriction sites. Regarding replication and delivery of RNA, the idea of viral replicons was discussed as an interesting topic to explore with experts. The decision to use the REPAIR system came after a detailed examination of other constructs that were discarded, as mentioned on our Engineering page.

Self-Reflection: Costs and timeframes delimited the diversity of designs we decided to test experimentally, as they were optimized according to existing literature, as great focus was made on the need to optimize the sequence across different reading frames. During these stages, the technical and academic focus of our meetings was evident, as we wanted to have a feasible and robust project before exploring broader applications.

Dr. David Hoerl

Postdoctoral Researcher - Human Biology and Bioimaging Chair, LMU

Summary: We contacted Dr. Hörl in the context of our Model work, reaching out to him due to his previous experience with codon optimization and fluorescence imaging, as well as his previous iGEM background. In the meeting, Dr. Hörl suggested the weighting of codon optimization functions, focusing on the Codon Adaptation Index (CAI) over reducing the GC content, which was the team’s approach until this point. Regarding exclusion parameters, he suggested applying high penalties instead of excluding sequences, especially for hidden stop codons. For analyzing the fluorescence readout, he suggested spectral unmixing tools to us, like Lumos, a plug-in for Image J. Finally, for secondary structure prediction, he encouraged us to use tools like Oligominer and Nupack to predict and account for off-frame motifs.

Self-Reflection: The meeting was valuable as it provided many insights from Dr. Hörl, bridging our theoretical knowledge with his practical experience on the design of bioinformatics tools. However, we also needed to consider the availability of resources for readout and how his suggestions would impact our intended flexibility of the algorithm.

Integration:

• His feedback was crucial in adjusting our codon optimization algorithm by increasing the weight of CAI over GC content, as well as applying penalties rather than exclusions. You can read more about this on our Model page.

Prof. Dr. Friedrich Simmel

Professor for Physics of Synthetic Biological Systems - TUM

Summary: Prof. Simmel’s research focuses on artificial molecular machines, a highly relevant topic to molecular recording technologies. Additionally, as a former PI of multiple iGEM teams, his advice was looked for to discuss our idea with someone who understands the time limitations, overall restrictions, and scope of iGEM projects. We discussed technical aspects mostly related to the CRISPR Cas and gRNA binding strengths, the timing of editing stages, and fast deaminating system requirements. Regarding the RNA aspect of our project, considerations like RNA survival time in cells or RNA triplex structures to increase stability. He also suggested exploring cell surface receptors for Cas placement and phase separation for counting in cellular droplets as possible experimental approaches.

Self-Reflection: The meeting with Prof. Dr Simmel was mostly focused on unforeseen technical aspects regarding the dynamics of RNA in cellular environments. Additionally, his focus on stability is something that stood with us throughout the project.

Integration:

• We agreed on Prof. Simmel’s advice to increase survival times and supply frequency of gRNAs. This also helped us realize RNA stability was a critical aspect for the efficient design of our tapes, which was further discussed in future sessions and meetings.
• After some team discussions, we concluded that aspects like cell surface receptors and RNA triplexes were an interesting approach but deemed too complex for our current project timeline.

Meeting with PI

Summary: The compatibility of XFPs was discussed, as the optimization of the XFPs and tapes design was deemed critical to be able to finish the project within its time frame. Advice was given to design positive controls, evaluate the use of aptamers (PP7), and contact experts on viruses to discuss gRNA expression and stability.

Self Reflection: The filter problem was a vital factor that hindered our research. The advice to achieve an optimal initial design implies a reliance on the predictability of algorithms in how experiments would work in the lab bench that initially delayed the beginning of wet-lab activities.

Prof. Dr. Thorben Cordes

Professor for Biophysical Chemistry - TU Dortmund

Summary: We approached Prof. Cordes for his expertise in single-molecule fluorescence spectroscopy, to comment on our approach to fluorescent protein selection and imaging techniques for in vivo measurements. We discussed the maturation time of fluorescent proteins and brightness, the use of narrow-band filters to avoid crosstalk, Fluorescence Lifetime Imaging Microscopy (FLIM), and RNA aptamers to have a faster biological readout.

Self-Reflection: The meeting had a technical focus, but proved very interesting to complement our manual approach to the fluorescent protein selection. We realized the use of RNA aptamers as an interesting topic we had not explored with much detail at first, but it became an important point In future meetings.

Integration:

• Following the meeting, as Prof. Cordes suggested, we decided to consider the maturation time of our XFPs over the brightness of their signals as a selection criterion.
• The potential use of RNA aptamers was extensively discussed and researched during several sessions, particularly as a potential readout method and for measuring basal expression levels. Ultimately, while we identified RNA aptamers as a feasible future option, we decided to focus on a translated RNA marker for our experimental work to ensure consistency with the use of XFPs.

Meeting with PI

Summary: This meeting was mainly targeted at presenting project updates, discussing options on XFP mutagenesis, efficiency through PP7/PCP, and options to measure basal levels of RNA-tape expression (XFP or aptamer). We decided, following REPAIR, to focus on a luciferase restoration assay to test tape switching. Additionally, we explored potential topics for a panel discussion on SynBio, ultimately choosing to focus on AI and machine learning as a contingent option for the field.

Self-Reflection: Time constraints pushed us to order the tape and troubleshoot possible - and inherent - problems that arose along the way. Regarding the panel, leaning towards AI was considered an option that would be more appealing to broader audiences than staying with a specific synbio topic, taking into account its already-widespread use in scientific practices.

Research Sessions

Summary: We explored options for the quantification of basal tape expression, and evaluated different potential XFPs for mutagenesis. Our research sessions also focused on upcoming education activities (more in the Education page) and the functions of the codon optimization algorithm, comparing it with an existing commercial option that does not work simultaneously in three reading frames.

Self-Reflection: We relied mainly on manual searches in FPbase databases and our previous knowledge of XFPs to explore options, and literature values for Kozak scoring. On non-technical aspects, the foundational aspect of our project made it challenging to explain our idea to possible sponsors and reach people outside academia. Additionally, we relied on connections related to previous iGEM Munich projects for our educational approaches, creating a continuity between different teams.

Kaiyi Jiang, PhD Candidate

PhD Candidate - Department of Biological Engineering, MIT

Summary: Kaiyi Jiang is a PhD candidate at the Gootenberg-Abudayyeh Lab at MIT, who are the first authors of REPAIR, the RNA-editing construct that we use in our project. The meeting intended to present the current state of our project to a scientist who works in the field of molecular recording, specifically with the REPAIR system. Kaiyi seemed very interested in our project, and we extensively discussed aspects of technical feasibility, gRNA design dependent on U6 promoter, and the importance of off-targets for therapeutic applications of molecular recording.

Self-Reflection: For us, this meeting was very relevant to understand how tools like REPAIR are used in scientific research. It provided us with a clear perspective on the practical challenges of using such technologies and the nuances of scaling up these technologies to biomedical applications.

Integration:

• Kaiyi emphasized the relevance of testing different tapes in our project. Due to time-economic reasons, we limited our experimental design to five tapes. However, taking his input, we developed a gRNA design algorithm to evaluate and optimize multiple gRNA designs. Although we generated numerous designs, we only tested the most promising candidates as determined by the algorithm. You can find more details about this process on our Engineering page.
• This meeting led us to evaluate different alternatives to Cas13 systems, such as LEAPER, HiFi or RFX. However, our independent literature review revealed that these options are not that suitable to our project, since our readout tape has very strict spatial and length requirements. Nonetheless, these alternatives were considered in future directions of our project.

Prof. Dr. Danny Nedialkova

Max Planck Research Group Leader and Professor for Biochemistry of Gene Expression - Max Planck Institute of Biochemistry and TUM

Summary: We reached out to Prof. Nedialkova, an expert in translation regulation and protein biogenesis, seeking advice on implementing fitness functions and scoring in our codon optimization tool. She encouraged us to consider weighted codon usage in our code, and that many of our feasibility concerns would be resolved by starting experimental work. Additionally, she made us aware of the impact of 5’ UTR on translation efficiency.

Self-Reflection: The proposal for using in vitro transcription for gRNA expression and electroporation as delivery methods were previously unconsidered approaches in our experimental design, leading to interesting discussions in future sessions. While IVT offered advantages, it diverged from our original in-vivo readout system design, also implying a high implementation cost of a technique that has not been established in our lab. Her advice to focus on wet-lab results showed our trust in predictive tools and differences in scientific practices that fostered constant self-reflection

Integration:

• Following Prof. Nedialkova’s advice, we implemented a strong 5’ UTR in our DNA tape design to optimize translation efficiency.
• Our codon optimization algorithm, which is available in the Model page, incorporated the suggestions from this meeting into their parameters.
• The recommendations on gRNA expression and delivery systems were heavily discussed in our next steps, while also being an essential component of a future implementation of progRAM as described in our Project Description.

Prof. Ulrike Protzer, M.D.

Professor for Virology and Director of the Institute of Virology - TUM and Helmholtz Zentrum München

Summary: As a virology expert, we contacted Prof. Protzer regarding our project’s replicon-based approach for gRNA delivery in aspects like feasibility, stability, replication mechanisms, and potential directions to explore in that regard. During the meeting, she highlighted the tradeoff between fluorescent protein overexpression toxicity and signal strength in replicon systems, suggesting some possible modifications.

Self-Reflection: A lot of the feedback Prof. Protzer regarding possible biomedical applications of our project, that while immensely useful, escaped a bit the foundational focus of our work. Nonetheless, they became relevant in the discussions regarding the applications of our project.

Integration:

• Her recommendation to contact Prof. Lohmann was pursued, as seen below in our reflection on that meeting.
• In the meeting, we were advised to pursue in-vitro transcription instead of replicons to avoid innate system inactivation, which was considered in our research sessions.

Meeting with PI

Summary: Discussing the input from previous interviews with experts was the main focus of this meeting. After careful evaluation, we decided to stay with the REPAIR system as we discussed how other alternatives that were proposed were not feasible with the length, spatial and structural requirements of our recording tape design. Nonetheless, we decided to do research and conceptualize how our system would work with other Cas constructs. PP7 was momentarily dropped due to inconclusive results, whereas in vitro transcription (IVT) for RNA delivery was not recommended due to the added experimental workload and high cost of recommended kits. Regarding our readout, we decided to focus on a fully translated RNA marker in our experimental design, using eUnaG to measure basal expression levels of our tape.

Self-Reflection: The foundational aspect of our project was determinant in our decisions during this meeting, as the focus on achieve feasibility and efficiency of the system was deemed as more relevant (as this state of the project) than off-targets, as we were not explicitly a therapeutic or biomedical tool. Additionally, options like PP7, Okra, eUnaG and avoiding IVT were decisions that were in line with the experimental setup and validated techniques in our lab.

Martin Grosshauser - PhD Candidate

PhD Candidate - Institute for Synthetic Biomedicine, Helmholtz Munich

Summary: We reached out to Martin Grosshauser, a PhD candidate within our PI’s group with experience in evolutionary algorithms and multi-frame codon optimization, for advice regarding the practical considerations of our codon optimization code and quality control mechanisms. He suggested evolutionary algorithm techniques to accelerate computing time and proposed seeding partially optimized sequences in the initial candidate population to reduce running time. He also shared with us his previous code for quality control using Splice AI, which could efficiently screen final generated sequences for uncommon splice sites.

Self-Reflection: At this point, the optimization tool had become a bottleneck to fully start our wet-lab activities, while its development has been hindered by the workload of team members involved in its design - factors that were critical in the avoidance of time-demanding techniques. Here, the use of Martin´s code heavily alleviated and accelerated the work by our Modeling team.

Integration:

• The Splice AI code was successfully used to quality control our sequences. This decision proved valuable as it helped us discover and manually remove a small number of potential splice sites that were not in our original exclusion list. More information on the use of these tools in our code is present in the Model page.
• Following Martin´s advice, we adapted the idea of seeding optimized sequences, not in the initial population, but by adding the best candidates from previous runs to newly initialized candidate populations, allowing us to continue previously finished runs effectively without artificially restricting the potential solution space.

Research Session

Summary: We discussed the outcomes of the codon optimization algorithm - which was constructed based on the feedback of previous meetings with experts - as well as possible cloning approaches to pursue.

Self-Reflection: Several factors influenced our cloning discussion before settling on Golden Gate, including previous experience with the technique, economic costs, shipping times, supplier length requirements and G/C content limitations.

Dr. Heiko Ott

Chief Business Officer / Chief Technology Officer - faceALS

Summary: As an advisor to past iGEM Munich teams,our meeting with Dr. Ott was vital to understanding the industry perspective, helping us explore potential applications and markets for our project, especially in regards to the fields of toxicology, developmental biology, conditional therapeutics and gene therapy. Since molecular recording is a novel field and we are working with the patented REPAIR technology, we aimed to assess market viability and identify potential commercial strategies. Dr. Ott. also highlighted the importance of responsible engagement with different publics and stakeholders about molecular recording technology.

Self-Reflection: The industrial perspective of Dr. Ott was key to obtain advise not restricted to academic settings. It also helped us undertand how markets approach emerging molecular biology technologies and how discussions about patents and IP are simultaneous to the development of a project.

Integration:

• After carefully considering his suggestions, his advice was key in our decision to postpone pursuing patents at this current state of the project, guided by financial and technical constraints.
• His feedback also speaks and corroborates the results of our survey on the public perception of molecular recording, which highlighted the need to engage with public concerns and expectations regarding molecular recording, as described in the respective section of this page.  

Prof. Dr. Volker Lohmann

Professor and Deputy Head of the Molecular Virology Department - Heidelberg University

Summary: The team reached out to Prof. Lohmann based on a recommendation from a colleague (Prof. Protzer) who was familiar with his work in viral systems and replicon technology. Our meeting centered around the implementation of viral replicons in our project, especially concerning aspects of RNA stability, replication rates, and interactions with cellular mechanisms. He highlighted potential challenges, such as cytotoxicity of alphavirus replicons, replication organelles hindering the accessibility of Cas-ADAR to modify the RNA and the time delay between viral RNA packaging and modification potentially interfering with the system’s performance.

Self-Reflection: The exchange with Prof. Lohmann helped us realize the scope of the challenge that was the use of replicons for gRNA delivery and expression, as the cytotoxicity of alphaviruses replicons became a major aspect that we were unaware of, even when it did not directly affect the biosafety levels required to work with them. Despite our extensive previous research, the meeting made us realize it was unfeasible within iGEM timelines to implement the replicon idea in a careful manner.

Integration:

• After discussing the insights provided by Prof. Lohmann, we decided to further research Alphavirus replicons as a method for gRNA expression and delivery only in the context of future implementations and not pursued experimentally. This decision was made to ensure that we fully understand the potential benefits and challenges of this approach instead of a quick and unsafe add-on to our project. Nonetheless, research on the topic continued and can be found in our Project Description page.
• Prof. Lohmann also helped us to adjust our experimental timeline in our proposed design of replicon use, as we agreed the viral replicon would need to be transfected 24 hours after the editor.
• The conversation on RNA vaccines and limitations of viral research also shaped the directions of our proposed applications, as these topics were heavily present in our own individual research on the topic.

Prof. Dr. Jay Shendure, M.D.

Professor for Genomic Sciences and Scientific Director - Seattle Hub for Synthetic Biology, University of Washington

Summary: We held a meeting with Prof. Shendure, an expert in molecular recording from the Seattle Hub for Synthetic Biology, one of the few institutes worldwide with dedicated molecular recording groups. The primary focus was to present our project and seek technical advice on RNA-based molecular recording, specifically compared to DNA-based tools. He gave us incredibly useful insights into how molecular recording speaks to the larger issues that limit biological measurements using conventional tools. He advised us to focus on the positive aspects the use of RNA brings to the field, like the possibility of scalability and recording events happening in faster timescales and without the limitations related to DNA-repair mechanisms.

Self-reflection: We were pleased to find that many of Prof. Shendure’s concerns were aspects we had already considered during our research, which was both validating and motivating. He also emphasized the importance of visual style, a self-explanatory and supportive use of diagrams, and greater macro-level narratives in scientific communication.

Integration:

• During our meeting, he mentioned difficulties and misunderstandings of our explanations of the project, despite his vast experience in molecular recording. Therefore, we realized more attention was needed into our engagement practices, without making previous knowledge assumptions when presenting a technology in such a novel field.
• Prof. Shendure gave us vital feedback on the visual presentation of our project, including some ideas for diagrams that we implemented in our homepage, Project Description and Project Promotion Video.
• Our conversation on applications of molecular recording that embrace the benefits of RNA guided the research directions we pursued and presented on our Project Description page.

Dr. Sierra K. Lear

Patent Agent - International Law Firm

Summary: We contacted Dr. Lear due to her vast experience with molecular recording in the Shipman Lab, as well as the valuable insights she could have from her current perspective as a patent agent. The meeting focused mainly on the difference between application-based research and more foundational projects like ours. Dr. Lear gave us critical recommendations on how to direct our future directions on the project, encouraging us to focus and embrace the transience of the RNA aspect of our system as one of its main advantages, along with valuable advice regarding the adequate timing to pursue IP rights regarding a novel project.

Self-Reflection: This meeting really showed us a focus on applications that were lacking in our project, as our “foundational focus” has pushed us a bit away from having those conversations and more into the academic/technical side. At the same time, it was helpful to reconsider the applications we were evaluating at that time, as those that required lineage tracing and long-lasting presence across generations seemed more suited for DNA molecular recording methods than our project.

Integration:

• We considered Dr. Lear´s recommendations in our proposal of possible applications of our project, which can be found on the Project Description page.
• Additionally, it prompted a team discussion on the need for patents and how that decision was unfeasible (or inconvenient) in the project’s current state.
• Finally, her advice to focus on the transience of RNA indirectly guides our current framing of the project in different presentations and events, highlighting this aspect as one of the most exciting characteristics of progRAM.

Meetings with PI

Summary: These meetings are intended to adjust research priorities in the final weeks of the project. It pushed our efforts on wet-lab deamination experiments towards XFPs rather than focusing on NLuc assays. Additionally, we explored previous ideas on PP7 aptamer, and we narrowed down the range of our possible applications discussed in our meetings with experts to explore with further research. More details on these outcomes can be found in our Results and Applications section in our Project Description page.

Self-Reflection: Most of the discussion leaned into technical aspects of our workflow and ways to obtain and present scientifically valid data with established techniques in our contextual laboratory setup, encouraging us to reflect on our analysis methods and the learnings that can be obtained from them.

Dr. Robert Meckin

Presidential Fellow - Social Statistics, University of Manchester

Summary: Dr. Meckin is a social scientist specializing in Science and Technology Studies (STS) with a focus on how synthetic biology practitioners, including iGEM teams, approach and make sense of their work. We contacted him to discuss our Human Practices work and the implications of self-reflective practices during our project. The meeting touched upon the interdisciplinary challenges between social and natural sciences, along with discussions on the meaning of iGEM both as a symbol and an established infrastructure in the SynBio field, understanding how the competition is pushing towards a specific imagined future where synthetic biology has gained widespread acceptance and adoption.

Self-Reflection: Dr. Meckin’s feedback helped us better understand and become critically aware how our project - and iGEM in general - is embedded in a broader socio-technical context within and beyond the SynBio field. It demonstrated that our reflective practices, such as STIR protocols, serve as an initial, modest acknowledgment of the inevitable external factors affecting our work and the work of others. And that recognizing these influences is crucial for engaging in responsible research practices.

Integration:

• The meeting with Dr. Meckin became a focal point of the conclusion of our Human Practices, as his advice for explicitly evidencing how our reflective and engagement work is integrated not only here but across all sections of our project and wiki - rather than a peripheral add-on.
• The conversation on the use of already-present metaphors and images in SynBio as a key enabler of interdisciplinary communication shaped our decision to present our construct according to standardized visual languages (SBOL Visual) in our Parts page and overall design choices.

Final Integration Session

Our iGEM project and the research directions we pursued were deeply shaped by the continuous feedback from experts, stakeholders, and our Principal Investigator (PI). Initially, our focus was on the technical feasibility of RNA-based molecular recording, exploring gRNA design and vector optimization. Expert insights, such as Dr. Hörl’s advice on codon optimization and Prof. Simmel’s focus on RNA stability, led us to refine our approach and help us with crucial components of our Model work.

Beyond the lab, our discussions with stakeholders like Prof. Shendure, Dr. Ott, and Dr. Lear broadened our perspective on the societal implications of molecular recording. They highlighted broader aspects of our project and contributed to our propositions of add-ons and future applications where we see our project being used. Finally, our use of the STIR framework and conversations with Dr. Meckin encouraged us to critically reflect on the ethical and societal dimensions of synthetic biology, understanding the responsibility and role of iGEM in shaping the future of synthetic biology.